DNA probes, method and kit for identifying antibiotic-resistant strains of bacteria

ABSTRACT

The invention relates to a method for identifying antibiotic-resistant strains of bacteria, especially strains of  Streptococcus pneumoniae.  According to the invention, the method is based on a combination of hybridization experiments using sensitivity-specific and resistance-specific probes. The invention also relates to the DNA probes and to a kit for carrying out the inventive method.

This application is a national stage filing under 35 U.S.C. §371 ofInternational Application No. PCT/DE98/01134 filed on Apr. 22, 1998.

The present invention relates to DNA probes, a method and a kit foridentifying antibiotic-resistant strains of bacteria.

The occurrence of antibiotic-resistant strains of bacteria, particularlyof streptococcus strains, represents an increasing problem. So far,antibiotic susceptibility tests have been carried out by isolatingbacteria and establishing a culture to define the minimum antibioticinhibitory concentration in a biological test. This method takes atleast 1 to 2 days. Well-calculated and thus optimum treatment is notpossible within this period. Therefore, there is a need for a fasteridentification of existing resistances.

The object of the present invention consists in providing products andmethods by means of which bacterial strains, particularly streptococcusstrains, can be tested fast and reliably for existing antibioticresistances.

This object is achieved by the subject matters defined in the claims.

The invention is described below by way of penicillin resistance ofStreptococcus pneumoniae. However, this principle also applies incorrespondingly general fashion to bacteria and resistances to otherantibiotics. Neisserias and MRSA strains (methicillin-resistantStaphylococcus aureus), which do not produce β-lactamase, are mentionedby way of example.

All of the penicillin-resistant S. pneumoniae strains have modifiedpenicillin target proteins (penicillin-binding proteins, PBP). The DNAsequences of genes which play a decisive part in the development ofpenicillin resistance in Streptococcus pneumoniae have meanwhile beendetermined in a number of penicillin-resistant streptococcus strains.Three genes were identified where differences between sensitive andresistant strains occur in connection with the development of penicillinresistance: PBP2x, PBP1a and PBP2b.

A comparison between the DNA sequences shows within the genes regionswhich are present in all of the sensitive S. penumoniae strains but aremodified in resistant strains. In this connection, reference is made toFIG. 1 which shows that the resistant strains differ more or lessmarkedly from the sensitive strain R6 in the PBP2x gene but also differamong themselves.

Because of the above finding that differences betweenpenicillin-sensitive and penicillin-resistant strains occur withincertain genes, the applicant developed DNA probes by means of whichresistant and sensitive strains can be differentiated. In thisconnection, reference is made to FIG. 4. The probes which are specificto sensitive sequences discriminate genes which code for low-affinityPBP variants responsible for penicillin resistance. The probes which arespecific to resistant sequences react with a very frequently occurringclass of PBP variants and can also be used for epidemiological purposes.

The applicant identified the following DNA probes [SEQ ID NOS.:1-8 ]:

a) Sensitivity-specific probes for PBP2x. The numerals in the column“nucleotide” refer to the nucleotides of the published sequence (Laibleet al., Mol. Microbiol. 5, pp. 1993-2002 (1991)). The numerals inparentheses refer to the codon and the position (1, 2 or 3) within thecodon of the structural gene. The number of bases in the nucleotide isgiven by “meric”.

Nucleotide (codon) oligonucleotide −meric 314-330 (105.2-110.3)AGT CAG CAA CGG GTA AG (1) 17 758-774 (253.2-258.3)AAC GAA CGA TGG ACG GT (2) 17 792-809 (264.3-270.2)CAT TTC CAG NCC CCT CCA (3) 18 (N: preferably C) 1098-1114 (366.3-372.1)TGC AGA TGC CAC GAT TC (4) 17 1302-1317 (434.2-439.3)CTG GTC AGC TTC CTG CG (5) 17 1677-1696 (559.3-566.1)TGG TTA TCT AGT CGG GTT AA (6) 20 1715-1731 (572.2-577.3)CTG TAT CGA TGA GTC CG (7) 17 2011-2029 (671.1-677.1)AAC AGT TCT GCT GAA GAA G (8) 19

b) Resistance-specific probes [SEQ ID NOS.: 14-17] for PB2x (as above;sequences in parentheses [SEQ ID NOS.: 20-23] are in accordance with thecorresponding sections of sensitive strains).

1065-1084 (AGG AGA AGT CTT TAA TAG T) (355.3-361.3) TGG AGA ATA NTT CAA TAG N (I) 19 (N: preferably C) 1202-1221 (CCC TCCTTG AGC AAA AGA TG) (401.2-407.3)  GTC TAC TTG AAC AAA AAA TG (II) 201549-1566 (TTG GTA GGG ACG GAT CCG) (517.1-522.3) TTA GTT GGG ACG GAC CCT (III) 18 1759-1776 (GTG ACG GTC CAA CAA CCT)(587.1-592.3)  GTA ACN NTT CAA CAG CCT (IV) 18 (N: preferably G)

c) Sensitivity-specific probes [SEQ ID NOS.: 9-12] for PBP1a (valuesrefer to the nucleotides of the published sequence of the structuralgene; Martin et al., EMBO J. 11, pp. 3831-3836 (1992))

(1034-1051) TAG GAG CAC GCC ATC AGT 18 (specific in most knownsequences) 1631-1648 GAC GAA ATG CCT ATC TTG 18 1722-1740 CTC TCA ATTTGT AGC ACC T 19 1794-1812 CTA TTC TAA CCG TCT GAC A 19

d) Resistant specific probes [SEQ ID NOS.: 18-19] for PBP1a ([SEQ IDNOS.: 24-25] in parentheses).

(TAC AGA CGA ATA CGT TGC C) 945-963  CTC CGA NCA ATA CGT CTC T 19 (N:preferably T) (GCA CCT GAT GAA CTA TTT GC) 1735-1754  GCT CCA GAT NAAATG TTT GT 20 (N: preferably G)

e) Sensitivity-specific probes [SEQ ID NO.: 13] for PBP2b (values referto the nucleotides of the published sequence of the structural gene;Hakenbeck, R., Matrin, C., Dowsen, C., Grebe, T., J. Bacteriol. 176, pp.5574-5577 (1996))

1329-1348 ATC AAA TAC CTA TAT GGT CC 20

N=any nucleotide

The above probes and those differing therefrom by one or severalnucleotides, preferably up to 4 nucleotides, respectively, are perfectlysuited to test unknown Streptococcus pneumoniae strains for resistanceto penicillin.

For this purpose, bacteria according to the invention are centrifugedoff a sample and in the case of S. pneumoniae the PBP genes (theresistance determinants) are amplified directly via PCR (polymerasechain reaction) as described in the literature (Grebe and Hakenbeck(1996), Antimicrob. Agents Chemother. 40, pp. 829-834). The advantage inconnection with S. pneumoniae consists in that a detergence-inducedlysis occurs rapidly and thus PCR can be carried out withoutlong-winding DNA preparations. Since this step fails with otherstreptococci, only pneumococcus DNA is amplified specifically by meansof this step. As an alternative, bacterial DNA (chromosomal and/orextrachromosomal) is isolated according to standard methods. This DNA ishybridized with at least one sensitivity-specific probe and with atleast one resistance-specific probe under standard conditions with whicha person skilled in the art is sufficiently familiar (see e.g. Maniatiset al., Molecular Cloning, Cold Spring Harbor, N.Y.: Cold Spring HarborLaboratory). The hybridization is preferably carried out under stringentconditions such as 20° C. below the melting point of the hybridizingDNA. The oligonucleotides are preferably chosen such that they havesimilar melting temperatures and thus several of them can be tested inthe same hybridization batch under the same conditions (see FIG. 2). Theoligonucleotides are preferably labeled when offered (P³², S³⁵,biotin/avidin system; dioxygenine (DIG)-labeled; fluorescein-labeled)and hybridized against immobilized DNA. As an alternative, theoligonucleotides are offered on an oligonucleotide microarray innon-labeled fashion and the DNA to be hybridized is obtained via PCR andlabeled while amplified.

It can be concluded from the hybridization result whether or not theunknown strain is sensitive to antibiotics. Depending on the resistancegene, at least one sensitivity-specific probe and oneresistance-specific probe should be used for the hybridization. However,the DNA of the unknown strain is hybridized advantageously with severalsensitivity-specific and resistance-specific probes in succession, sinceevaluation of resistance by means of only one combination ofsensitivity-specific probes and resistance-specific probes can beinaccurate and rather only serve as a rough estimate. This appliesparticularly to the case of penicillin resistances in pneumococci andneisserias.

Preferred hybridization conditions depend on the AT content and lengthof the oligonucleotides. The person skilled in the art can selectsuitable conditions on the basis of his technical knowledge. Thus, e.g.10-100 ng/ml labeled oligonucleotide for PBP2x (see above) are used inSSC hybridization solution at a hybridization temperature of 45°-60° C.for at least 5 hours, preferably overnight.

The oligonucleotides can also be used as PCR primers to as to develop aPCR test therewith (see FIG. 3). This test can dispense with thesomewhat more time-consuming hybridization. However, several PCRs mustbe used per strain. This method is suitable above all forepidemiological purposes.

The circumstance that less probes are known for PBP1a and particularlyfor PBP2b follows from the fact that smaller gene regions are ofsignificance for resistance in PBP1a and particularly in PBP2b andtherefore also only smaller regions have a sequence variation.

The invention also relates to a kit for carrying out the above method.This kit comprises means for isolating DNA from bacteria and for the PCRamplification of specific resistance determinants, respectively,sensitivity-specific DNA probes and resistance-specific DNA probes(lyophilized and as oligonucleotide microarray, respectively), reagents,solutions, buffers and means for hybridization and the subsequentdetection of hybridized DNA. The sensitivity-specific DNA probes andresistance-specific DNA probes are preferably the ones listed above.

The advantage of the present invention is that bacteria, particularlypneumococci, can be assessed as to antibiotic resistance within theshortest time, i.e. within few hours. This enables subsequently awell-calculated and efficient treatment of diseased patients.

The invention is further described by means of the figures showing:

FIG. 1 shows a comparison of gene sections of the Streptococcuspneumoniae PBP2x gene between penicillin-sensitive andpenicillin-resistant strains; codon 85-750 R6: penicillin-sensitivestrain Others: penicillin-resistant strains

FIG. 2 shows the hybridization on an oligonucleotide array Thearrangement of the probes on the array is indicated in the first blockof the figure. Numerals (1) to (8) and (I) to (IV), respectively,correspond to the numbering of the above-mentioned probes for PBP2x.

A) Strain R6, a sensitive S. pneumoniae laboratory strain andrepresentative of other sensitive strains: all sensitivity-specificoligonucleotides (Nos. 1-8) are identified whereas all of the fourresistance-specific oligonucleotides (I-IV) are not identified.

B) Strain 2349 whose PBP2x gene belongs to a frequently and globallyoccurring class of PBP2x genes of resistant pneumococci. Only one of thesensitivity-specific oligonucleotides is identified, since the modifiedsequence does not cover the 3′ region of the gene. All of the othersensitivity-specific oligonucleotides (Nos. 2-8) do not hybridize. Allof the resistance-specific oligonucleotides (I-IV) hybridize.

C) Strain J19, a resistant strain having a PBP2x which only in part hassequences which correspond with that of strain. 2349. One of theresistance-specific oligonucleotides (III) does not respond.

D) Strain Pn12, a resistant strain from Papua, whose PBP2x has anunusual sequence. Five of the sensitivity-specific oligonucleotides donot respond, an evidence for the fact that the PBP2x has no continuoussensitive sequence (and thus conveys resistance). However, theresistance-specific oligonucleotides do not respond either, whichindicates that an unusual sequence is also present in the “resistant”gene region. Strains like this one are an exception but can be detectedclearly on account of screening, above all when further oligonucleotidesare used which are specific to other PBPs.

FIG. 3 shows the result of PCR reactions for the amplification of S.pneumoniae R6 DNA as an application on an agarose gel. The PCR primersused were the above PBP2x probes marked above by (1) to (7) as forwardprimers and probe (8) as reverse primer each. PCRs having probes (I) asforward primer as well as (IV) as reverse primer and (II) as forwardprimer as well as (IV) as reverse primer, respectively, were carried outas a control. M=size marker. It can be clearly identified that on thegel shown only the sensitivity-specific probes result in anamplification whereas none takes place with resistance-specific probes.

FIG. 4 (A-HH) Detection of the probes according to the invention bysequence comparisons.

The invention is further described by means of an example.

EXAMPLE Isolation of S. pneumoniae Bacterial DNA and Subsequent Testingfor Existing Resistance to Penicillin

Bacteria of the strain S. pneumoniae R6 are inoculated in brain-heartinfusion (BHI) broth and allowed to grow at 37° C. overnight. The cellswere centrifuged off and lyzed by, resuspension of the sediment in 10 μlof 10 mM Tris/HCl buffer, pH 7.2, 0.05% triton-X100. 1 μl of the cellsuspension each are used per 20 μl PCR batch (0.2 μl Taq polymerase, 1pM oligonucleotide primer each, 2 μl 10×PCR buffer, 4-6 mM MgCl₂). 25cycles with 5 seconds of annealing at 96° C., 5 seconds of annealing at52° C., 10 seconds of extension at 72° C. suffice for the PCR reaction.

A) Agarose Gel Electrophoresis

The following primer combinations are used in the PCR reactions(conditions see above):

Forward primers reverse primers probe (1) probe (8) probe (2) probe (8)probe (3) probe (8) probe (4) probe (8) probe (5) probe (8) probe (6)probe (8) probe (7) probe (8) probe (I) probe (IV) probe (II) probe (IV)

The designations of the probes correspond to the numerals for PBP2x,indicated above in connection with the sequences.

In each case, 4 μl aliquots of the PCR reactions were applied onto a1.5% agarose gel and separated electrophoretically. The result is shownin FIG. 3. It follows therefrom that R6 is a sensitive strain.

B1) Dot Blot

S. pneumoniae R6 bacterial DNA is amplified with common primers (Grebeand Hakenbeck (1996), Antimicrob. Agents Chemotherap. 40, pp. 829-834)in a PCR reaction (conditions see above). The PCR-amplified DNA isdenatured by heating (2 min. at 96° C., then 4° C)., 2 μl thereof eachare applied per sample onto a nylon membrane. The DNA is fixed byirradiation with long-wave U.V. light onto the membrane, andnon-specific binding sites are saturated at 60° C. in prehybridizationsolution (6×SSC, 5×Denhardt solution, 0.1% SDS, 50 mM Na phoshphatebuffer, pH 6.5, 0.1 mg/ml heringsperm DNA) with mild shaking for 5hours. The hybridization with the PBP2x sensitivity-specificoligonucleotide probes (1) to (8) and the resistance-specific probes (I)to (IV), respectively, is carried out in hybridization buffer (likeprehybridization solution but with 50 ng/ml oligonucleotide probe) atabout 50° C. overnight. The filter is washed at room temperature with2×SSC/0.1% SDS at 55° C. for 2×5 minutes. The samples are stained usinganti-DIG-AP conjugate in accordance with the instructions from themanufacturer (Boehringer Mannheim). Here, it also turns out that onlythe sensitivity-specific probes result in a hybridization, whichindicates that the S. pneumoniae strain R6 is a penicillin-sensitivestrain.

B2) Oligonucleotide Microarray

The method is carried out as defined above under B1) but with thedifference that the oligonucleotides are offered as finished array andthe DNA to be hybridized must be labeled via PCR by means of DIG orfluorescein-labeled nucleotides. The principle of high-densitymicroarray hybridization is described in “Nature Biotechnology 14, pages1675-1680, 1996”. The result of this experiment is shown in FIG. 2A.

25 1 17 DNA Streptococcus pneumoniae 1 agtcagcaac gggtaag 17 2 17 DNAStreptococcus pneumoniae 2 aacgaacgat ggacggt 17 3 18 DNA Streptococcuspneumoniae misc_feature (10) Nucleotide at position 10 is n wherein n isany nucleotide but preferably c. 3 catttccagn cccctcca 18 4 17 DNAStreptococcus pneumoniae 4 tgcagatgcc acgattc 17 5 17 DNA Streptococcuspneumoniae 5 ctggtcagct tcctgcg 17 6 20 DNA Streptococcus pneumoniae 6tggttatcta gtcgggttaa 20 7 17 DNA Streptococcus pneumoniae 7 ctgtatcgatgagtccg 17 8 19 DNA Streptococcus pneumoniae 8 aacagttctg ctgaagaag 19 918 DNA Streptococcus pneumoniae 9 taggagcacg ccatcagt 18 10 18 DNAStreptococcus pneumoniae 10 gacgaaatgc ctatcttg 18 11 19 DNAStreptococcus pneumoniae 11 ctctcaattt gtagcacct 19 12 19 DNAStreptococcus pneumoniae 12 ctattctaac cgtctgaca 19 13 20 DNAStreptococcus pneumoniae 13 atcaaatacc tatatggtcc 20 14 19 DNAStreptococcus pneumoniae misc_feature (10)..(19) Nucleotides atpositions 10 and 19 are n wherein n is any nucleotide but preferably c.14 tggagaatan ttcaatagn 19 15 20 DNA Streptococcus pneumoniae 15gtctacttga acaaaaaatg 20 16 18 DNA Streptococcus pneumoniae 16ttagttggga cggaccct 18 17 18 DNA Streptococcus pneumoniae misc_feature(6)..(7) Nucleotides at positions 6 and 7 are n wherein n is anynucleotide but preferably g. 17 gtaacnnttc aacagcct 18 18 19 DNAStreptococcus pneumoniae misc_feature (7) Nucleotide at position 7 is nwherein n is any nucleotide but preferably t. 18 ctccgancaa tacgtctct 1919 20 DNA Streptococcus pneumoniae misc_feature (10) Nucleotide atposition 10 is n wherein n is any nucleotide but preferably g. 19gctccagatn aaatgtttgt 20 20 19 DNA Streptococcus pneumoniae 20aggagaagtc tttaatagt 19 21 20 DNA Streptococcus pneumoniae 21 ccctccttgagcaaaagatg 20 22 18 DNA Streptococcus pneumoniae 22 ttggtaggga cggatccg18 23 18 DNA Streptococcus pneumoniae 23 gtgacggtcc aacaacct 18 24 19DNA Streptococcus pneumoniae 24 tacagacgaa tacgttgcc 19 25 20 DNAStreptococcus pneumoniae 25 gcacctgatg aactatttgc 20

What is claimed is:
 1. A method for testing Streptococcus pneumoniae forresistance to penicillin, the method comprising the steps of: a)isolating DNA from a Streptococcus pneumoniae strain, b) hybridizing theDNA obtained in step (a) with at least one sensitivity-specific DNAprobe and at least one resistance-specific DNA probe, and c) determiningwhether or not said Streptococcus pneumoniae strain is sensitive topenicillin or not by detecting which probe or probes hybridize. whereinthe sensitivity-specific probes are selected from the group of sequencesconsisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,SEQ ID NO: 5, SEQ ID NO: 6 and sequences which differ from saidsequences by one to four nucicotides, wherein SEQ ID NOS.: 1-6 are,respectively: AGT CAG CAA CGG GTA AG, AAC GAA CGA TGG ACG GT, CAT TTCCAG NCC CCT CCA, TGC AGA TGC CAC GAT TC, CTG GTC AGC TTC CTG CG, and TGGTTA TCT AGT CGG GTT AA;

wherein N is any nucleotide.
 2. A method for testing Streptococcuspneumoniae for resistance to, penicillin the method comprising the stepsof: a) isolating DNA from a Streptococcus pneumoniae strain b)hybridizing the DNA obtained in step (a) with at least onesensitivity-specific DNA probe and at least one resistance-specific DNAprobe, and c) determining whether or not said Streptococcus pneumoniaestrain is sensitive to penicillin or not by detecting which probe orprobes hybridize, wherein the resistance-specific probes are selectedfrom the group of sequences consisting of SEQ ID NO: 14, SEQ ID NO: 15,SEQ ID NO: 16, SEQ ID NO: 17, and sequences which differ from saidsequences by one to four nucleotides, wherein SEQ ID NOS.: 14-17 are,respectively: TGG AGA ATA NTT CAA TAG N, GTC TAC TTG AAC AAA AAA TG, TTAGTT GGG ACG GAC CCT, and GTA ACN NTT CAA CAG CCT;

wherein N is any nucleotide.
 3. The method according to claims 1,wherein the probes are labeled radioactively.
 4. A method for testingStreptococcus pneumoniae for resistance to penicillin, the methodcomprising the steps of: a) isolating DNA from a Streptococcus pneumoniastrain, b) hybridizing the DNA obtained in step (a) with at least onesensitivity-specific DNA probe and at least one resistance-specific DNAprobe, and c) determining whether or not said Streptococcus pneumoniaestrain is sensitive to penicillin or not by detecting which probe orprobes hybridize; wherein the sensitivity-specific probes are probeswhich specifically hybridize to the DNA of antibiotic sensitive strainsand are selected from the group of sequences consisting of SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 andsequences which differ from said sequences by one to four nucleotideswherein SEQ ID NOS.:1-6 are, respectively: AGT CAG CAA CGG GTA AG, AACGAA CGA TGG ACG GT, CAT TTC CAG NCC CCT CCA, TGC AGA TGC CAC GAT TC, CTGGTC AGC TTC CTG CG, and TGG TTA TCT AGT CGG GTT AA;

 wherein N is any nucleotide; and wherein the resistance-specific probesare probes which specifically hybridize to the DNA of antibioticresistant strains and are selected from the group of sequencesconsisting of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:17, and sequences which differ from said sequences by one to fournucleotides wherein SEQ ID NOS.:14-17 are, respectively: TGG AGA ATA NTTCAA TAG N, GTC TAC TTG AAC AAA AAA TG, TTA GTT GGG ACG GAC CCT, and GTAACN NTT CAA CAG CCT;

 wherein N is any nucleotide.
 5. A method for testing Streptococcuspneumoniae for resistance to penicillin, the method comprising the stepsof: a) isolating DNA from a Streptococcus pneumonia strain, b)hybridizing the DNA obtained in step (a) with at least onesensitivity-specific DNA probe and at least one resistance-specific DNAprobe, and c) determining whether or not said Streptococcus pneumoniaestrain is sensitive to penicillin or not by detecting which probe orprobes hybridize; wherein the sensitivity-specific probes are probeswhich specifically hybridize to the DNA of antibiotic sensitive strainsand are selected from the group of sequences consisting of SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6,wherein SEQ ID NOS.:1-6 are, respectively: AGT CAG CAA CGG GTA AG, AACGAA CGA TGG ACG GT, CAT TTC CAG NCC CCT CCA, TGC AGA TGC CAC GAT TC, CTGGTC AGC TTC CTG CG, and TGG TTA TCT AGT CGG GTT AA;

 wherein N is any nucleotide; and wherein the resistance-specific probesare probes which specifically hybridize to the DNA of antibioticresistant strains and are selected from the group of sequencesconsisting of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO:17, wherein SEQ ID NOS.:14-17 are, respectively: TGG AGA ATA NTT CAA TAGN, GTC TAC TTG AAC AAA AAA TG, TTA GTT GGG ACG GAC CCT, and GTA ACN NTTCAA CAG CCT;

 wherein N is any nucleotide.
 6. The method of claim 4, wherein the DNAisolated in step (a) is obtained from a strain of bacteria havingunknown antibiotic sensitivity or resistance.
 7. The method of claim 6,wherein step (b) is performed at least a second time, therebyhybridizing the DNA obtained in step (a) with at least a seconddifferent sensitivity-specific DNA probe and at least a second differentresistance-specific DNA probe.
 8. The method of claim 5, wherein the DNAisolated in step (a) is obtained from a strain bacteria strain havingunknown antibiotic sensitivity or resistance.
 9. The method of claim 8,wherein step (b) is performed at least a second time, therebyhybridizing the DNA obtained in step (a) with at least a seconddifferent sensitivity-specific DNA probe and at least a second differentresistance-specific DNA probe.
 10. The method of claim 1, wherein theDNA isolated in step (a) is obtained from a strain of Streptococcuspneumoniae having unknown penicillin sensitivity or resistance.
 11. Themethod according to claim 2, wherein the probes are labeledradioactively.
 12. The method of claim 2, wherein the DNA isolated instep (a) is obtained from a strain of Streptococcus pneumoniae havingunknown penicillin sensitivity or resistance.